304 



J. HOPE, J. SIKORSKI AND C. S. WHEWELL 



Table 1. The examination of potential methods for isolating pigment-containing granules 



in keratinous materials. 



Reagent 



Details of treatment 



PH 



Material 



Sodium hydroxide 

 2.5 A' 

 3.0^ 

 5.0^ 



Sodium carbonate (15 %) 



Sodium sulphide (0.5 M) 



Sodium bisulphite (0.1 A') in 50/50 

 //-propyl alcohol/water 



Monothioglycol (0.2 M) in 50/50 

 77-propyI alcohol/water 



Thioglycollic acid (0.2 M) in 50/50 

 //-propyl alcohol/water 



Sodium thio-glycollate (0.5 M)with 

 sodium hydroxide 



Monothioglycol (0.5 M) with 

 sodium hydroxide 



Sodium bisulphite (0.3 M) 

 and urea (10.0 M) 



Monothioglycol (0.5 M) and 

 ammonium thiocyanate (9 M) 



Hydrochloric acid (6 A') 



PHT reagent (see text) 



Boiling for 5 min. 

 At 98 C for 3 hr. 



Boiling for 24 hr. 



Boiling for 45 min. 



Refluxing for 24 hr. 

 Refluxing for 48 hr. 



Refluxing for 24 hr. 



Refluxing for 24 hr. 



Refluxing 3 to 24 hr. 

 Refluxing 3 J hr.'- 

 Refluxing for 4i hr. 



Refluxing for 24 hr. 



Refluxing for 24 hr. 



Refluxing for 24 hr. 



Boiling for 48 hr. 

 Refluxing for 24 hr. 



BWM. 



1 C/R— Crow or Rook; Ch.— Chicken; G— Grouse; BWM.— Black Welsh Mountain Wool. 



2 Washed 3 times in boiling 2 N hydrochloric acid for few minutes. 



containing granules involved treatment with kera- 

 tinolytic reagents. In the present investigation the 

 suitability of several reagents to isolate pigment- 

 containing granules, from black feathers and dark 

 brown Welsh Mountain Wool, was examined. 



These not only provided data on the effectiveness 

 of the reagents as a means of isolating granules but 

 also yielded information on the relative resistance 

 of the materials under different courses of treatment. 

 The reagents used and other relevant data are sum- 

 marized in Table 1. 



The experimental procedure was as follows: the feath- 

 ers were washed in warm tap water and soap solution, 

 and after being dried were purified by successive refluxing, 

 for 24 hours, in diethyl ether and ethyl alcohol; finally 

 they were washed in ten changes of distilled water. The 

 quill and rachis were cut ofT and discarded, so that only 

 web was used in the subsequent work. 



One tenth gram of purified feathers, or hairs, were 

 treated in 10 ml of reagent. After extraction (see Table 1) 

 suspensions were filtered through sintered glass filter and 

 centrifuged. Washing was carried out by centrifuging in: 

 (i) three changes of extracting liquor, (ii) three changes 

 of distilled water, (iii) three changes of acetone and, 

 finally, (iv) two changes of diethyl ether. Residues were 

 then stored under vacuum over phosphorus pentoxide. 



For electron microscopy, residues were re-suspended 

 by gentle grinding in a drop of distilled water on a micro- 

 scope slide, using a glass stirring rod. A drop of the 

 suspension was deposited on a supporting nitrocellulose, 

 or carbon, film on the electron microscope grids and 

 dried over phosphorus pentoxide. 



From the above experiments the following main 

 conclusions can be drawn. The residues obtained 

 from all treatments, except that involving the PHT 

 reagent, were either contaminated by keratinous 

 material (see fig. 1 ) or showed varying degrees of 

 attack on the pigment-containing granules. Generally 

 the amounts of keratinous contaminants varied only 

 slightly from one reagent to another with perhaps 

 one exception of treatment (for longer periods) in 

 sodium thioglycollate, which was capable of yielding 

 relatively clean pigment-containing granules. Fur- 

 ther improvement in this respect was obtained, by 

 additional washing for a few minutes in 2 A^ hydro- 

 chloric acid. However, in view of the very high pH 

 values involved in the original reagent, more detailed 

 investigations are necessary before an answer can 

 be given about the applicability of this method, 

 particularly since greater electron transparency was 



